Technologies for causing expression of various proteins in cells are essential today, with function analysis of cells and proteins in cells, production of useful proteins, and the like in wide use. In order to cause expression of specific proteins within cells, a method of transfecting a gene into the cells is useful, and a method in which a plasmid vector is employed as a carrier of the gene to be transfected is widely used. A plasmid is a double-stranded, annular structure that exists within cytoplasm of a bacterium or yeast cell and autonomously performs replication independently of chromosomal DNA. In the field of genetic engineering, a plasmid that has undergone various modifications and had foreign genes inserted therein is transfected into a cell as a plasmid vector and is used in gene expression.
Many plasmid vectors primarily include replication origin points for replicating in a host cell of E. coli bacteria and the like; a promoter sequence; a terminator sequence (some also being referred to as a poly(A)-binding sequence or poly(A) sequence); a multicloning site that includes various restriction enzymes for inserting a drug resistant or auxotrophic marker gene or a foreign gene; and the like. The promoter sequence is a sequence controlling initiation of gene transcription. In many cases, sequences of approximately 500 to 1000 nucleobases are used. In order to be optimized for gene expression using a plasmid vector, these sequences often employ a promoter having a large amount of gene expression, such as a virus, or are modified from an original promoter sequence. Further, the terminator sequence is a sequence that controls termination of gene transcription, or that stabilizes transcribed RNA. In many cases, sequences of approximately 200 to 1000 nucleobases are used.
The simplest general example of a plasmid vector structure is as follows (FIG. 1): (1) a target RNA-expressing DNA sequence is amplified using a PCR method by employing a primer that includes a restriction enzyme site, then an amplified product of the target RNA-expressing DNA sequence is obtained that includes the restriction enzyme site on both ends thereof; (2) the amplified product obtained in step (1) and the plasmid vector are processed by the restriction enzyme; (3) a restriction enzyme processing product of step (2) is purified; (4) the plasmid vector and amplified product of step (3) are connected by a ligation reaction and rendered annular; (5) a ligation reaction product of step (4) undergoes transgenesis into E. coli bacteria, then is dispensed in a plate that includes a selected medical agent and is incubated overnight at 37° C.; (6) a colony of E. coli bacteria is taken from the plate of step (5), then the E. coli bacteria is cultivated in a liquid culture medium; (7) the plasmid vector is purified using the E. coli bacteria of step (6), then a sequence, structure, and the like of the resulting plasmid vector is confirmed; and (8) the E. coli bacteria having the target plasmid vector confirmed in step (7) are cultivated, then the plasmid vector is purified to obtain a necessary amount thereof. Typically, completing all of these steps requires five days to a week, or even longer. It can be said that the process is one of the experiments acting as a rate limit on a project's progress.
The plasmid vector is advantageous in that the plasmid vector is capable of maintaining comparative stability even when inside a transfected cultured cell, and of obtaining a high level of RNA expression. However, the structure of the plasmid vector requires a great deal of time for enzyme processing and growth of the E. coli bacteria, which requires time to complete, as noted above. Moreover, technical skill is required. Further, even the step of amplifying the constructed plasmid vector within the E. coli bacteria takes one night for the plasmid vector to undergo transgenesis into the E. coli bacteria and to form a colony. Culture of the E. coli bacteria that includes the plasmid vector takes from twelve hours to (typically) about one night, and amplification of the plasmid vector also takes time. Therefore, a method for gene expression is sought that is technically simple and greatly compresses the time required for construction and amplification of DNA for expression.
In recent years, due to the discovery of a polymerase with a high degree of accuracy, synthesis of genes in a few hours using a PCR method has become possible. Thus, instead of an annular plasmid vector that takes time and effort to produce, a method is considered in which a linear form of linear DNA amplified using the PCR method is transfected as-is into a cell to perform gene expression. When a gene for transfection produced with this method is capable of achieving sufficient gene expression within a cell, conventional plasmid construction requiring time and effort can be swapped for fast and easy production of linear DNA using the PCR method. The amount of time taken can thus be greatly compressed and development of a high level of through-put can be anticipated. However, there is a problem that even when the linear DNA produced by the PCR method undergoes gene transfer to a cell, an amount of expression is markedly lower as compared to a plasmid vector given the same genetic sequence, or expression does not occur.
Patent Literature 1 recites a method of preparing a DNA fragment using the PCR method, the DNA fragment including a promoter sequence, a target gene, an expression marker gene, a terminator sequence, and a polyadenylation signal sequence, which are sequences required for expression in cells. However, in order to resolve the problem that linear DNA is likely to degrade within the cell and that a high level of expression is difficult to obtain, the method of Patent Literature 1 adopts a technique of making the above-noted DNA fragment annular, and transfects an annular plasmid vector into the cells in a manner similar to conventional methods. Accordingly, time and effort required for making the DNA annular, selecting the DNA made annular, and the like is equivalent to the conventional time and effort, and is thus not capable of compressing the time and effort to any marked degree in comparison to conventional methods.
Patent Literature 2 teaches a linear DNA as a linear expression element, the linear DNA including a promoter, a coding region, and the like. A method described in Patent Literature 2 individually amplifies each structural element (such as the promoter), then, by annealing of single-stranded DNA overhanging terminals ends of each, the structural elements are non-covalently bonded together. In order to do this, a method using a dUMP-containing PCR primer and uracil-DNA glycosylase, a method using a non-basic phorphoramidate, a method using rU/RNaseA, and the like can be employed. However, there is a problem that all of the methods require high-cost reagents and cumbersome manipulation, and so they cannot be said to be methods for gene expression that enable application to high-throughput, that are technically simple, and that greatly compress time. Patent Literature 3 teaches a method for simple production of linear DNA fragments for gene expression in a cell-free system, the linear DNA fragments including a promoter and a terminator on a plasmid vector. However, Patent Literature 3 is unable to resolve the low level of gene expression using linear DNA in a cell culture.
Further, Non-patent Literature 1 teaches a method for expressing a plurality of genes, each having a different expression amount, using a single vector by including, on the same vector, a poly(A) signal of SV40 having a modified AATAAA sequence downstream of one target gene and including a poly(A) signal of SV40 not having the modified AATAAA sequence downstream of another target gene. In addition, Patent Literature 4 recites a method for amplifying protein production by modifying a nucleotide sequence of an untranslated region of DNA composed of an untranslated region that includes, in order, a coding region, a translation stop codon, and a polyadenylated signal, the nucleotide sequence being modified such that a distance between the translation stop codon and an AATAAA polyadenylated signal is 300 base pairs or less, then using the nucleotide sequence in a vector DNA. However, each of these methods makes use of an annular plasmid vector and increases the time and effort to produce the vector more than conventional methods. Accordingly, at present, no method has been discovered for gene expression using linear DNA that enables a high level of gene expression that rivals a plasmid vector in a cell culture, that is technically simple, and that greatly compresses the time required for production.